This years major accomplishments are in the following areas: 1) Temporal coupling between specifications of neuronal and macular fates of the inner ear Understanding the molecular pathways which distinguish between neuronal and hair cell fates during inner ear development have long-term therapeutic implications. The information gained could provide insight into how to tweak neural stem cells as a source for hair cell replacement. In the past year, we published a combined cell lineage and transplantation study on the neurogenic region of the developing chicken inner ear using a lipophilic dye. We showed that after auditory and vestibular neuroblasts have exited from the neurogenic region of the otic cup, the neurogenic epithelium develops into the vestibular sensory organs, maculae of the saccule and utricle, respectively. By switching the medial-lateral axis of the neurogenic region surgically, we showed that the vestibular neurons acquire their identity prior to their delamination from the otic epithelium. More importantly, the fate of utricular macula appears to be fixed concomitantly at the time of transplantation as the vestibular neurons. These results indicate that specification of the neural and sensory fates of the inner ear are temporally coupled and further suggest that the two fates may share the same molecular pathway at some point of their development. 2) Transcription factor Emx2 controls stereocilia polarity and line of polarity reversal formation in maculae of the inner ear and neuromasts of the lateral line Emx2 encodes a homeodomain transcription factor, which is required for formation of multiple organs such as the brain, olfactory epithelium and urogenital system. In the inner ear, it was reported that the lack of Emx2 affected stereocilia polarity pattern in the maculae of the inner ear. Normally, the maculae of the utricle and saccule of the inner ear, responsible for detecting linear acceleration, exhibit a line of polarity reversal (LPR), across which stereocilia are arranged in a mirror-image pattern. Our previous gain- and loss-of-Emx2 function results indicate that Emx2 is necessary and sufficient to reverse stereocilia polarity of sensory hair cells in the inner ear. The expression domain of Emx2 dictates the position of the LPR in both maculae. During the past year, we have extended our studies to investigate whether the cell autonomous function of Emx2 applies to other sensory organs that exhibit LPR such as neuromasts of the lateral line in zebrafish. Two types of neuromasts are present in the zebrafish lateral line based on stereocilia orientation pattern: anterior-posterior (A-P) and dorsal-ventral (D-V) oriented neuromasts. Emx2 is only expressed in hair cells that share the same polarity, which constitute half of the hair cells within a given neuromast. Only the posterior or ventral pointing stereocilia in the A-P and D-V neuromast express emx2, respectively. We generated gain- and loss-of emx2 mutant fish and the results are entirely consistent with the effects of Emx2 in the mouse inner ear. In the gain of function fish, all the stereocilia are pointing towards the posterior or ventral direction in the A-P and D-V neuromasts, respectively. Consistently, in the loss-of-function fish, stereocilia are pointing towards the anterior or dorsal direction in the respective A-P and D-V neuromasts. Based on these results, we propose that Emx2 has a conserved role in controlling stereocilia polarity in all sensory hair cells. 3) Role of retinoic acid in the specification of striola and central zone of the vestibular sensory organs The striola and central zone of the vestibular sensory organs are specialized regions comprised of Type I hair cells innervated by calyceal nerve endings. These specialized hair cells and neurons have been proposed to be essential for mediating vestibular reflexes which have quick response time of milliseconds. The molecular mechanisms underlying the specification of these regions during development are not known. We found that a retinoic acid (RA) synthesizing enzyme, Raldh3, is expressed in the developing maculae and cristae except in the presumptive striola and central zone, respectively. In contrast, one of the retinoic acid degradation enzymes, Cyp26b1, is expressed in the striola and central zone. Based on these expression patterns, we propose that formation of the striola and central zone require low level of retinoic acid, whereas the rest of vestibular sensory organs require higher RA levels. We are currently testing this hypothesis using in vitro cultures as well as generating mouse mutants with gain and loss of RA functions. Our long-term goal is to generate mouse mutants that lack the striola and central zone to address the functions of these specialized regions. 4) Lineage analysis of Sonic Hedgehog expressing cells in the spiral ganglion of the mouse The organ of Corti, the sensory epithelium for detecting sound in mammals, is tonotopically organized such that sensory hair cells at the base are most sensitive to high frequency sounds and their counterparts at the apex are most sensitive to low frequency sounds. The molecular mechanisms that give rise to the structural basis for the tonotopy such as the shape of the hair cell and the size and height of the stereocilia bundle on the apical surface of the hair cell, are not clear. Notably, an unusual developmental feature of these sensory hair cells is thought to be related to the tonotopy establishment. Namely, hair cell precursors undergo cell cycle exit starting from the apex of the cochlear duct and progresses towards the base, whereas hair cell differentiation starts promptly at the base after terminal mitosis and progresses towards the apex. Previous results from our lab show that the delay in apical hair cell differentiation is due to restricted expression of Sonic Hedgehog (Shh) emanating from the spiral ganglion (SG) near the cochlear apex. The loss of Shh in the developing SG causes a shorten cochlear duct and premature hair cell differentiation at the apex. Thus, deciphering the regulation of Shh expression in the SG is an important factor in understanding cochlear formation. In the past year, we investigated whether the restricted Shh expression by the cochlear apex represents a unique subpopulation of neurons or it represents a certain stage of neuronal development that all SG neurons undergo. To distinguish between these possibilities, we conducted a lineage study by crossing Shh-creER mice with a cre reporter strain, Rosa-tdTomato, and activated cre at different times of development using tamoxifen. We then compared tdTomato-positive cells in the SG of Shh-creER; Rosa-tdTomato ears. In contrast to the restricted Shh expression pattern during cochlear development, majority of the SG neurons are tdTomato-positive indicating that they are derived from the Shh lineage. These results suggest that the restricted Shh expression detected during inner ear development represents a stage of neuronal development rather than a unique subpopulation of neurons. We are currently conducting experiments to correlate the timing of cell cycle exit with the onset of Shh expression in the developing SG. In the long run, the nature and regulation of this transient Shh expression will be investigated.